Many intracellular and secreted signaling molecules are synthesized as precursors that undergo elaborate post-translational processing events critical to their normal and pathological functions. To better understand these events, we are studying the biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor, an extracellular signaling peptide that is prenylated and carboxyl methylated. Mature a-factor is derived from a precursor that terminates with a CaaX motif ("C" is Cys, "a" is aliphatic, "X" is any of several residues). The biogenesis of the a-factor precursor is distinctive, involving: (1) C-terminal (CaaX) processing (by the farnesyl transferase Ram1p/Ram2p, the endoproteases Ste24p and Rcelp, and the methyltransferase Ste14p), (2) N-terminal proteolytic cleavage (by the endoproteases Ste24p and Axllp), and (3) a non-classical export mechanism (mediated by the a-factor exporter, Ste6p). We have recently shown that one of these components, Ste24p, is a multispanning membrane protein with intrinsic protease activity that acts in dual steps of a-factor biogenesis, mediating both C- and N-terminal processing. Furthermore, in extending our studies to mammalian systems we discovered a new mammalian Ste24p substrate, prelamin A (the nuclear lamin A precursor). In this proposal we will focus on the activity, topology, and interaction of the membrane-associated a-factor CaaX processing components (Ste24p, Rcelp, Ste14p) and explore the possibility that these proteins (and others) may form a CaaX processing "machine" (Aims 1-3). CaaX processing renders a-factor extremely lipophilic; yet to exit the cell a-factor must transit through the aqueous environment of the cytosol, and thereafter must diffuse through the extracellular milieu. We will examine the trafficking of a-factor, both inside and outside of the cell, and genetically identify novel factors involved in these processes (Aims 4-6). Essentially all of the steps in a-factor biogenesis are mediated by multispanning membrane proteins. Therefore in the long-term, our studies will lead to a better fundamental understanding of the structure, function, catalytic properties, and organization of multispanning membrane proteins that mediate diverse cellular processes, extending beyond a-factor biogenesis. Such processes include the proteolytic processing events that generate the cholesterol biosynthesis regulator (SREBP), the Alzheimer's precursor protein (APP), and mammalian prelamin A (defects in which result in human laminopathies). Our studies also bear on the development of chemotherapeutic agents designed to inhibit the CaaX processing of oncogenic Ras proteins.